The neuro-and vasculoprotective properties of estrogen have been amply demonstrated in laboratory investigations. However, clinically, there are indications that hormone replacement therapy in women may not be beneficial, and even detrimental. The present project is based upon the concept that clinically-relevant circumstances may occur where estrogen replacement therapy (ERT) is no longer neuroprotective, but neurotoxic. One such circumstance is diabetes/chronic hyperglycemia (CH). Recent results showed that, whereas chronic "physiologic" ERT, in non-diabetic, ovariectomized (OVX) rats protects the brain against ischemic damage, ERT in diabetic OVX females exacerbates the damage. This project is designed to delineate the mechanisms behind this "transformation". The rationale derives from the parallel observations that CH promotes an increase in the levels of advanced glycation end-products (AGEs), while ERT promotes an increased expression of the AGE receptor, RAGE, on cerebrovascular endothelial cells. The resultant increase in RAGE activation unleashes a cascade leading to a sustained increase in the activity of the pro-inflammatory transcriptional regulator, NFkB, and a heightened potential for cerebral inflammatory activity following an ischemic insult. The 4 specific aims of this proposal are guided by the following hypotheses: 1) ERT in diabetic (streptozotocin-treated, chronically [>1 mo] hyperglycemic) OVX females will exacerbate, while ERT in non-diabetics will attenuate post-ischemic (transient forebrain ischemia) cerebrovascular inflammation, as reflected by the extent to which leukocytes adhere to pial venules. 2) Blocking RAGE, its ligands (e.g., AGE), or its downstream effector, NFkB, either via antisense or pharmacologic inhibitor treatments, will prevent the pro-adhesive action of ERT in diabetics. 3) Preventing leukocyte adhesion (via leukopenia), in addition to AGE, RAGE or NFkB blockade, will prevent ERT-associated exacerbation of ischemic brain damage. 4) Chronic progesterone (P) replacement, when combined with ERT, will prevent the pro-inflammatory and neurotoxic actions seen with ERT alone in the diabetic OVX female. Results will be evaluated based upon whether the females are intact, OVX, or OVX + 17B-estradiol (E2)-treated (or E2+P-treated), and whether the rats are diabetic or non-diabetic. The principal variables to be monitored will be post-ischemic (0-10h reperfusion) pial venular adhesion of rhodamine-6G-labeled leukocytes; neuronal cell loss (at 72h reperfusion); and expression of key proteins. The established technique being used for quantitating leukocyte adhesion involves chronic placement of closed cranial windows, intravital microscopy/videometry, and computer-assisted image processing. The results of these studies will permit us to gain an understanding of some of the mechanisms behind estrogen's conversion to the "dark side" and should lead to improvements in the clinical efficacy of hormone replacement treatments. [unreadable] [unreadable]